Introduction
The cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is one of the most economically important pests throughout the world (Ebert and Cartwright, Reference Ebert and Cartwright1997) and is responsible for severe yield losses both through direct feeding and indirect virus transmission in various crops (Guncan et al., Reference Guncan, Madanlar, Yoldas, Ersin and Tuzel2006; Wumuerhan et al., Reference Wumuerhan, Guo, Ma, Gao, Zhang, Zhang and Ma2019; Zhang et al., Reference Zhang, Chen, Shan, Dong, Shi and Gao2020). Controlling cotton aphids in China is challenging due to its high reproductive capability, large population, tolerance and environmental adaptability (Eid et al., Reference Eid, El-Heneidy, Hafez, Shalaby and Adly2018). However, the indiscriminate and long-term chemical application poses an environmental risk and results in high levels of insecticidal resistance (Wu and Guo, Reference Wu and Guo2005). In addition, widespread insecticide resistance in A. gossypii hinders chemical control (Zeng et al., Reference Zeng, Pan, Song, Li, Lv, Gao, Tian, Peng, Xu and Shang2021; Cheng et al., Reference Cheng, Li, Chen, Ni, Lv, Liang, Guo, Zhen, Liang and Gao2023). Therefore, new pest control strategies are urgently needed, such as targeting specific genes that can block pest development. This is important in contemporary pest management programmes to delay the development of insecticide resistance in cotton aphids.
RNAi has been considered a novel tool that promotes eco-friendly pest management strategy. Some researchers have investigated the mechanism of pest resistance to insecticides, including Helicoverpa armigera, Ectropis oblique, and A. gossypii using RNAi (Pan et al., Reference Pan, Wen, Chen, Gao, Zeng, Liu, Tian and Shang2020; Zheng et al., Reference Zheng, Wu, Huang, Zhang and Qiu2024). To explore novel control methods, some researchers had conducted studies on the growth and development of insects using RNAi to select appropriate target genes. Knockdown ferritin genes (NlFer1 and NlFer2) led to retarded growth and 100% mortality in Nilaparvata lugens nymphs (Shen et al., Reference Shen, Chen and Zhang2021). Silencing ApisCHS led to mortality and moulting rate of Acyrthosiphon pisum was 44% and 51.3% after 72 h compared with dsGFP group, respectively (Ye et al., Reference Ye, Jiang, An, Yang, Shang, Niu and Wang2019). The knockdown of CHS1 caused up to 43%, 47%, and 59% mortality in 3th instar A. gossypii after feeding dsCHS1 for 24, 48, and 72 h, respectively (Ullah et al., Reference Ullah, Gul, Wang, Ding, Said, Gao, Desneux and Song2020). These studies suggested that insect growth and development genes can be used as target genes for RNAi to achieve effective pest control.
In insects, 20-hydroxyecdysone (20E) and juvenile hormone (JH) are the key hormones in regulating various development and reproductive processes (Jindra et al., Reference Jindra, Palli and Riddiford2013; Yamanaka et al., Reference Yamanaka, Rewitz and O'Connor2013). JH is one of the most critical sesquiterpenoid hormones, which plays various roles in the regulation of essential physiological processes, including moulting, metamorphosis, reproduction, diapause and migration (Riddiford et al., Reference Riddiford, Hiruma, Zhou and Nelson2003; Zhao et al., Reference Zhao, Zhou, Li, Cai and Hua2017; Li et al., Reference Li, Jia and Li2019; Xu et al., Reference Xu, Deng, Mu, Fu, Guo and Li2019; Riddiford, Reference Riddiford2020; Oi et al., Reference Oi, Ferreira, Silva, Bienstman, Nascimento and Wenseleers2021; Zhang et al., Reference Zhang, Li and Liu2022a). Some studies have revealed that juvenile hormone acid methyltransferase (JHAMT) is a rate-limiting enzyme in the JH synthesis pathway (Kinjoh et al., Reference Kinjoh, Kaneko, Itoyama, Mita, Hiruma and Shinoda2007; Marchal et al., Reference Marchal, Zhang, Badisco, Verlinden, Hult, Van Wielendaele, Yagi, Tobe and Vanden Broeck2011; Daimon and Shinoda, Reference Daimon and Shinoda2013; Cai et al., Reference Cai, Tao, Zhao, Xia, He and Wang2022). RNAi-mediated silencing of JHAMT in insects causes growth disorders, reduced reproductive quality, and diapause (Yin et al., Reference Yin, Qiu, Huang, Tobe, Chen and Kai2020; Tian et al., Reference Tian, Guo, Li, Zhu, Liu and Wang2021). In Tribolium castaneum, RNAi was performed on TcJHAMT3 in 3rd instar larvae, causing early pupation and significantly smaller adults than that of the control group (Minakuchi et al., Reference Minakuchi, Namiki, Yoshiyama and Shinoda2008). Furthermore, the mortality of Leptinotarsa decemlineata larvae fed dsJHAMT1 and dsJHAMT2 was 30.0% and 32.2%, respectively, while 66% and 62% of surviving larvae failed to pupate (Fu et al., Reference Fu, Li, Zhou, Meng, Lu, Guo and Li2016). Silencing the JHAMT gene decreased larval growth rate, higher larval mortality, pupation of fewer larvae and fewer adult emergence (Navale et al., Reference Navale, Manamohan, Asokan, Krishna, Sharath, Prasad, Latha, Krishna and Ellango2017). These results suggested that JHAMT played an important role in insect growth and development. Our previous studies showed that feeding transgenic cotton lines expressing dsAgCYP6CY3 (the TG cotton) not only increased the susceptibility of cotton aphids to neonicotinoid insecticides, but also delayed the development of cotton aphids (Zhang et al., Reference Zhang, Wei, Wei, Liu and Liu2022b).
To elucidate the molecular events underlying the physiological changes in cotton aphids that fed on the TG cotton, we selected the JHAMT gene that response to the TG cotton based on transcriptome sequencing analysis of cotton aphids in this study. Then the expression pattern of AgJHAMT during the nymph stages and after AgCYP6CY3 gene silencing were detected, respectively. Subsequently, the gene function was analysed by spraying-mediated RNAi combined with the methoprene rescue experiment. This study laid a foundation for further investigation of the mechanism of the TG cotton delayed the development of cotton aphids and helped to evaluate its potential for developing novel control strategies against this pest.
Materials and methods
Insects
The susceptible cotton aphid's population was collected in 2010 from the Anningqu Town in Urumqi, Xinjiang province, China. They were reared on cotton seedlings (Gossypium hirsutum) under 25 ± 1 °C and relative humidity of 50–60% with a 16 h L: 8 h D photoperiod in the Xinjiang laboratory of biological resources and genetic engineering of Xinjiang University. The newborn nymphs (<12 h) were used in RNAi and methoprene rescue experiments.
Transcriptome sequencing (RNA-Seq)
The newborn nymphs were released on the non-transgenic cotton (the NT cotton) and the transgenic cotton lines expressing dsAgCYP6CY3 (the TG cotton), respectively. The cotton seedlings were covered with plastic cups to prevent the aphids escaping. Each treatment had three biological replicates. Then the total RNA of cotton aphids that fed on the NT cotton and the TG cotton for 36 h were extracted and used to detect the relative expression of CYP6CY3 in cotton aphids, transcriptome sequencing and transcriptome verification experiments. Total RNA was extracted using TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China) following the manufacturer's instructions. The quality and concentration of RNA were confirmed using agarose gel electrophoresis and NanoDrop-1000 Spectrophotometer (Thermo Scientific, CA, USA), respectively. Part of the total RNA was sent to Biomarker Technologies Co., Ltd. (Beijing, China) for library preparation. The other part was reverse transcribed into cDNA using TransScript All-in-one First-Strand cDNA Synthesis Supermix for qPCR (One-Step gDNA Removal) kit (TransGen Biotech, Beijing, China) following the manufacturer's instructions, and cDNA templates were stored at −80 °C for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis to verify the reliability of transcriptome results.
Multiple sequence alignments and phylogenetic analysis
The AgJHAMT gene was cloned. Briefly, the PCR procedures were as follows: initial pre-denaturation at 94 ℃ for 5 min followed by 35 cycles of 94 ℃ for 30 s, 56 ℃ for 30 s, and 72 ℃ for 1 min, and a final elongation step at 72 ℃ for 10 min. Target amplicons were purified, then products were transferred to vector pMD19-T (TakaRa, Dalian, China) for sequencing. The AgJHAMT sequence was analysed using Primer Premier 5 and DNAMAN. Other JHAMT protein sequences were obtained from the National Center for Biotechnology Information (NCBI). The phylogenetic tree was constructed with the neighbour-joining method based on 1000 bootstrap replicates using MEGA 10.0. The primers used in this study were shown in table 1.
Table 1. Primer sequences
Note: The underlined sequence is the T7 promoter sequence added at the 5’end of the primer.
Expression pattern of AgJHAMT in A. gossypii
To study the effect of the TG cotton on the growth and development of cotton aphids, we detected the relative expression level of AgJHAMT in cotton aphids that fed on the TG cotton. In addition, we detected the expression pattern of AgJHAMT in cotton aphids that 8 hours before and 8 hours after each moulting peak as each instar's early and late stages, respectively. The total RNA and cDNA of A. gossypii were obtained according to the above method. RT-qPCR was performed on the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster city, CA, USA). The experiment was conducted with 3 independent biological replicates, each with 2 technical replicates. The relative expression level was calculated using the 2−△△Ct method (Livak and Schmittgen, Reference Livak and Schmittgen2001), and the 18S rRNA was used as the internal control.
Synthesis of double-stranded RNA (dsRNA)
We truncated the coding sequence of the AgJHAMT gene as the interference fragment, and synthesised dsRNA according to the MEGAscriptTM RNAi kit (Ambion, Huntingdon, USA). The dsRNA was analysed by 1% agarose gel electrophoresis and quantified using NanoDrop-1000 spectrophotometer. Green fluorescent protein dsRNA was synthesised under identical conditions and was used as a control. The specific primers of two dsRNA fragments were shown in table 1.
RNA interference (RNAi)
The silencing efficiency of AgJHAMT and its effects on the growth and development of cotton aphids were investigated following our previous method (Wei et al., Reference Wei, Zhang, Liu, Gao and Liu2021). Synthetic dsGFP and dsJHAMT (the final concentration is 500 ng/μL), approximately 200 μL per plant were directly sprayed on the cotton seedlings containing the newborn nymphs using a 2 mL volume sprayer from four directions, respectively. The dsRNA was sprayed only once during the entire experiment. The cotton aphids fed on the leaves were directly exposed to dsRNA, and the aphids continuously fed on dsRNA-sprayed cottons. The plastic cups covered cotton seedlings to prevent aphids from escaping. The sprayed H2O and dsGFP were used as control groups, respectively. Each treatment was repeated 3 times.
After RNAi, cotton aphids were collected at 2nd instar, 3rd instar, 4th instar and A1 (adult 1st day) from the sprayed H2O, dsGFP control groups and the sprayed dsJHAMT treatment group to detect the relative expression level of AgJHAMT gene using RT-qPCR according to the above method.
After the newborn nymphs were treated with dsRNA, the number of moults, deaths and newborn progeny nymphs were recorded and then removed until all treated adult aphids died. The life table was constructed for aphids using data from the study described above. Life table parameter calculation formula: net reproductive rate: R0 = Σ(lxmx), the mean generation time: T = Σ(xlxmx)/Σ(lxmx), the intrinsic rate of increase: rm = (lnR0) / T, finite rate of increase: λ = exprm, population doubling time: td = ln2/rm = 0.6931/rm, x represents age in days, lx represents the age-specific survival rate, mx represents the age-specific fecundity, lxmx represents age-specific maternity.
Rescue assay by methoprene (juvenile hormone analogue, JHA) treatment
A rescue assay was conducted to study the effects of methoprene on cotton aphids after AgJHAMT silencing. The newborn nymphs were sprayed with dsJHAMT for 1.5 days, then sprayed with methoprene (0.01 μg/μL). The experiment was conducted with three independent biological replicates.
Statistical analysis
Statistical analyses were performed using SPSS 20.0. Significant differences between the three groups were calculated using one-way analysis of variance (ANOVA) in conjunction with Tukey's test, and different letters were used to indicate significance at P < 0.05, while Student's t-test were used to analyse pairs of groups (*P < 0.05, **P < 0.01, ***P < 0.001).
Results
Screening AgJHAMT by transcriptome sequencing analysis
In the current study, high-quality base reads were obtained; all the reads and base counts and their qualities were listed in table S1. In total, 87.52 Gb of clean data were obtained. The sequencing data had an average GC content of 38.34% and >94.80% of each sample had a quality score of Q30. The efficiency of comparison between reads and reference genomes ranged from 90.13% to 91.33%. We selected 15 differentially expressed genes for RT-qPCR and compared them with transcriptome sequencing analysis, indicating that the RNA-Seq results were reliable (fig. S1). The primers used for verification were listed in table S2.
Compared with the cotton aphids that fed on the NT cotton, 151 differentially expressed unigenes (DEGs) were found in the cotton aphids fed on the TG cotton, which included 51 up-regulated genes and 100 down-regulated genes (fig. 1a). In the KEGG analysis, 151 DEGs were assigned to 47 KEGG pathways, and the top 20 significantly enriched KEGG pathways were shown in fig. 1b. Of these, 13 were involved in nutrient metabolism, such as sugar metabolism (4), insect hormone biosynthesis (1), amino acid related metabolism (1), and lipid metabolism (7). Three pathways were related to metabolic detoxification: drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450 and lysosome. According to the GO terms, DEGs were divided into three categories (biological processes, molecular functions, and cellular components) containing 42 variety classes. Catalytic activity (50), binding (38), and single-organism processes (40) contained the most UniGenes in the three categories (fig. 1c). According to the above transcriptome data analysis results, we found JHAMT involved in insect hormone biosynthesis was up-regulated expression in cotton aphids which fed on the TG cotton, and it was selected to explore its function in A. gossypii.
Figure 1. Function annotation and enrichment of DEGs. (a) volcano plot of differentially expressed genes of A. gossypii fed on the TG cotton (red spots represent significantly up-regulated genes; blue spots represent significantly down-regulated genes). (b) the most enriched KECG pathways of A. gossypii after fed on the TG cotton. (c) GO function annotation analysis of A. gossypii which fed on the TG cotton.
Cloning and sequence analysis of AgJHAMT
Using cDNA from adult cotton aphids as template, an 801 bp ORF sequence of a JHAMT orthologue (AgJHAMT) was amplified by PCR and then sequenced. A comparison of the amino acid sequences of five JHAMTs indicated that the putative SAM-binding motif is well conserved in all methyltransferases (fig. 2). Then the phylogenetic tree was constructed with JHAMT from A. gossypii and other insect species by MEGA 10.0. It clustered the AgJHAMT protein in a well-supported Hemiptera clade (fig. 3). This result demonstrated that AgJHAMT gene had been cloned and it was closely related to the JHAMT of other Hemiptera.
Figure 2. Multiple alignments of amino acid sequences of JHAMT in four insect species. Identical residues are indicated with black backgrounds; high homology residues are indicated with blue backgrounds. The red dotted box represents the SAM-binding motif. The details and GenBank accession numbers of the six JHAMTs are listed in the order illustrated: A. gossypii JHAMT (XP_027843037.2); Aphis glycines JHAMT (KAE9531301.1); Aphis craccivora JHAMT (KAF0764091.1); Rhopalosiphum maidis JHAMT (XP_026813805.1); and Rhopalosiphum padi JHAMT (WJN62156.1).
Figure 3. Phylogenetic analyses of AgJHAMT. The phylogenetic tree is based on amino acid sequences using the neighbor-joining method with a bootstrap of 1000 through MEGA 10.0. The numbers at the branches’ nodes represent the bootstrap support level for each branch.
Analysis of the expression pattern of AgJHAMT
In addition, we investigated the expression pattern of AgJHAMT in cotton aphids that fed on the TG cotton. The results showed that the relative expression level of AgJHAMT in the TG group was significantly higher than that of the NT group at 24 h, 48 h, 72 h and 96 h, respectively. It was twice as high as that of the NT group at 96 h (fig. 4). This result suggested that a high expression level of AgJHAMT of cotton aphids that fed on the TG cotton might lead to its developmental delay.
Figure 4. The relative expression level of AgJHAMT in A. gossypii which fed on the TG cotton. NT: A. gossypii which fed on the NT cotton; TG: A. gossypii which fed on the TG cotton. * Indicates a significant difference between the NT group and the TG group (mean ± SE, n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001, Student's t-test).
The temporal expression profile of AgJHAMT was examined using RT-qPCR analysis. The results showed that AgJHAMT was expressed during the nymph stages of cotton aphids, and its relative expression increased with development. The relative expression level in the early stages of each instar was significantly higher than that of the corresponding late stages (fig. 5). This result showed that the expression level of AgJHAMT fluctuated with instars, implying that the function of this gene was related to the developmental period of A. gossypii.
Figure 5. The relative expression level of AgJHAMT in different developmental stages of A. gossypii. E: early stage of nymphs; L: late stage of nymphs. * Indicates a significant difference between the early stage and the late stage (mean ± SE, n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001, Student's t-test).
Effect of silencing AgJHAMT on the growth and development of A. gossypii
Cotton aphids were collected at 2nd instar, 3rd instar, 4th instar and A1 from control and treatment groups to detect the silencing efficiency of target gene (fig. 6). The result showed that the relative expression level of AgJHAMT was no significance between the sprayed H2O and dsGFP control groups. With the development of the aphids, the target gene expression level was significantly lower in the dsJHAMT-treatment group than that of the sprayed H2O and dsGFP control groups (P < 0.05), respectively. The relative expression level of AgJHAMT decreased by 66.2%, 42.9%, 67.8% and 43.6% in the 2nd instar, 3rd instar, 4th instar nymph and A1 compared with the sprayed H2O control group, respectively, indicating that the expression level was effectively silenced by spraying dsJHAMT.
Figure 6. The relative expression level of AgJHAMT in A. gossypii. Different letters indicate statistically significant differences (mean ± SE, n = 3, P < 0.05, Tukey's HSD test).
To further explore the role of AgJHAMT in the growth and development of cotton aphids, the newborn nymphs were sprayed with synthetic dsJHAMT. The result showed that there were four peaks in the frequency distribution of the number of nymphs moulting, which corresponded to the four developmental stages of cotton aphids (fig. 7). The generation duration of cotton aphids treated with dsJHAMT was 4 days, while 4.5 days durations were observed for the sprayed H2O and dsGFP control groups. The overall developmental period of cotton aphids in the treatment group was 0.5 days earlier than that of the two control groups. This result suggested that the developmental period of cotton aphids was advanced after AgJHAMT silencing.
Figure 7. Developmental period of A. gossypii sprayed with dsJHAMT.
Mortality and reproduction hadbeen recorded daily intervals to assess the population dynamics of cotton aphids following the application of dsJHAMT. There had a weak effect on the death and fecundity of cotton aphids (fig. S2). Based on the above data, the effects of AgJHAMT silencing on the growth and development of cotton aphids were further investigated. We constructed a life table to evaluate various population parameters of cotton aphids after spraying dsJHAMT (table 2). The results showed that the T and t values of cotton aphids in dsJHAMT treatment group (6.88 ± 0.15) (1.65 ± 0.06) were significantly shorter than that of the sprayed H2O control group (7.6 ± 0.14) (1.97 ± 0.09) (P < 0.05), respectively. These results suggested that silencing the AgJHAMT gene shortened the mean generation time and population doubling time of cotton aphids, disrupted its growth and development.
Table 2. Life table parameters of cotton aphids sprayed with dsJHAMT
Note: The data in the table are mean ± SE; different lowercase letters in the same row indicate that there is a significant difference at the 0.05 level between different treatments.
Methoprene (juvenile hormone analogues, JHA) rescues the effect of dsJHAMT
The aphids growth were significantly delayed following RNAi-mediated silencing the AgJHAMT. A rescue assay with methoprene was performed to investigate whether the lack of JH caused this result. The developmental period of aphids was recorded. The four moulting times of cotton aphids in dsJHAMT treatment group were shorter than those of the H2O control group. In the JHA rescue group, the first moulting time (before rescue) of nymphs treated with dsJHAMT was 0.5 day earlier than that of the sprayed H2O control group, and then one of the two groups that sprayed dsJHAMT was sprayed JHA (0.01 μg/μL) to rescue. The second moulting time (the 1st moulting time after rescue) of nymphs was still 0.5 day earlier than that of the sprayed H2O control group. But the third moulting time (the 2nd moulting time after rescue) of nymphs was longer than that of the dsJHAMT group, which was coinciding with the 3rd moulting time from the H2O control group, and then the next moulting time of nymphs was the same as the 4th moulting time from the H2O control group (fig. 8a). These above results suggested that methoprene (JHA) could do rescue the rapidly developmental period of cotton aphids caused by AgJHAMT silencing.
Figure 8. Effects of methoprene rescue on growth and development of A. gossypii. (a) Developmental period. (b) Age-specific survival rate (lx).
In addition, the age-specific survival rate (lx) result showed that both the lx curve of cotton aphids in the sprayed dsJHAMT and JHA rescue treatment groups had a tendency to decrease compared with that of the H2O control group, respectively (fig. 8b). And the life cycle of the cotton aphids in the H2O, dsJHAMT and JHA rescue treatment groups was 25, 20, and 24 days, respectively. The life cycle of the cotton aphids in the sprayed dsJHAMT treatment group was 5 days earlier than that of the sprayed H2O control group. The rescued group using methoprene was similar to the sprayed H2O control group. These results indicated that the life cycle of cotton aphids was advanced by silencing AgJHAMT, which was rescued though treatment with JHA.
Discussion
Transcriptome sequencing is used to identify the key genes and pathways linked with the growth and development of the insect pest. For example, cheng et al., used transcriptome sequencing to screen the HNF gene affecting embryonic development and egg hatching in N. lugens (Cheng et al., Reference Cheng, Li, Li, Song, Zeng and Lu2020). The transcriptome sequencing was used to select and knock out the gene EcRA associated with insect moulting, which affects Spodoptera exiguais mortality and the ecdysone signalling pathway (Zhang et al., Reference Zhang, Ma, Ma, Hu, Wang, Song, Ren and Ma2021). In insects, ecdysteroids and sesquiterpenoid hormones of arthropods play vital roles in regulating various developmental processes such as moulting, growth, and metamorphosis (Daimon et al., Reference Daimon, Kozaki, Niwa, Kobayashi, Furuta, Namiki, Uchino, Banno, Katsuma, Tamura, Mita, Sezutsu, Nakayama, Itoyama, Shimada and Shinoda2012; Yamanaka et al., Reference Yamanaka, Rewitz and O'Connor2013; Mirth et al., Reference Mirth, Tang, Makohon-Moore, Salhadar, Gokhale, Warner, Koyama, Riddiford and Shingleton2014). The titres of these two hormones are precisely coordinated by biosynthesis and metabolism pathways to regulate the physiological and developmental processes. Although ecdysteroids initiate the moulting process, JH determines the nature of the moulting (Lenaerts et al., Reference Lenaerts, Van Wielendaele, Peeters, Vanden Broeck and Marchal2016).
As an important gene in insect JH biosynthesis, JHAMT affects the physiological processes of insect growth, development and reproduction (Navale et al., Reference Navale, Manamohan, Asokan, Krishna, Sharath, Prasad, Latha, Krishna and Ellango2017; Zhou et al., Reference Zhou, Zhang, Yang, Yuan, Wang and Dou2022). JHAMT was characterised in several insect species, including Holcocerus hippophaecolus (Zhang et al., Reference Zhang, Wang, Zhou, Li, Luo, Weng and Zong2016) and T. castaneum (Xu et al., Reference Xu, Yan, Qian, Chen, Guo, Zhu, Wu and Chen2022), closely related to their growth and development. In addition, RNAi-mediated silencing of JHAMT in Bactrocera dorsalis greatly decreased the JH III titre, affecting the body length and overall size of larvae (Zhou et al., Reference Zhou, Zhang, Yang, Yuan, Wang and Dou2022). The H. armigera pupation was reduced following the silencing of JHAMT (Jaiwal et al., Reference Jaiwal, Natarajaswamy and Rajam2020). The expression changes of JHAMT affects the JH titre, thus disrupting the growth and development process of insects. Other studies had shown that the developmental expression profile of JHAMT in Drosophila melanogaster correlates with changes of the JH titre (Niwa et al., Reference Niwa, Niimi, Honda, Yoshiyama, Itoyama, Kataoka and Shinoda2008). The research showed that the content of JH titre increased in the early 4th instar, while decreasing in the later age of the 4th instar. The JH titre was sharply increased in the early 5th instar. Similarly, the content of JHAMT gene was also reduced in larvae (Kinjoh et al., Reference Kinjoh, Kaneko, Itoyama, Mita, Hiruma and Shinoda2007). Our study found that the relative expression of AgJHAMT in the early stages of each instar was significantly higher than that of the corresponding late stages. Therefore, we speculated that the fluctuation of AgJHAMT expression with instar maybe due to the influence of JH titres. It also implied that AgJHAMT was closely related to the developmental period of A. gossypii.
Generally, functional genes involved in insect development or key metabolic processes could be suitable for RNAi targets (Kola et al., Reference Kola, Renuka, Madhav and Mangrauthia2015; Yu et al., Reference Yu, Liu, Huang, Chen, Sun, Duan, Ma and Xia2016). JHAMT is a specific target for developing new insect growth regulators or insecticides because it regulates JH synthesis in insect development and reproduction (Hiruma and Kaneko, Reference Hiruma and Kaneko2013). Studies have shown that the body length and the overall size of B. dorsalis larvae after silencing JHAMT were significantly decreased and reduced (Zhou et al., Reference Zhou, Zhang, Yang, Yuan, Wang and Dou2022). Other studies have shown that compared with the dsGFP control, the lower level of JHAMT1 expression leads to reproductive arrest of cabbage beetles (Tian et al., Reference Tian, Guo, Li, Zhu, Liu and Wang2021). In this study, transcriptome data analysis results showed that JHAMT was up-regulated in the insect hormone metabolic pathway directly related to the growth and development of cotton aphids. The study also showed that overexpression of AgJHAMT gene in cotton aphids after fed on the TG cotton (fig. 2). These results indicated that the developmental retardation of cotton aphids by the TG cotton might be contributing to the up-regulation of AgJHAMT. Additionally, the developmental period of cotton aphids was advanced after RNAi-mediated silencing of AgJHAMT, compared with the sprayed H2O control group, rm and λ of cotton aphids were also significantly increased, T and t were significantly decreased. These results further clarify our previous research (Zhang et al., Reference Zhang, Wei, Wei, Liu and Liu2022b). However, the influence of cumulative mortality and cumulative reproduction were subtle in the dsJHAMT treatment group compared with the sprayed H2O and dsGFP control groups, the results showed that the cumulative mortality was not significantly increased consistently by spraying dsJHAMT, but its cumulative reproductive was significantly increased from 5th to 9th days (P < 0.05) (Fig. S2). The reason for this result might be related to the concentration of juvenile hormone in A. gossypii. The appropriate concentration of hormones or analogues to regulate insect life activity is important, the effects caused by high or low concentrations of hormones are different or opposite (Staal, Reference Staal1975, Reference Staal1986; Champlin and Truman, Reference Champlin and Truman1998; Orth et al., Reference Orth, Lan and Goodman1999). It is reported that different concentrations of juvenile hormone analogues (0.1 μg/2μL, 1 μg/2μL, 5 μg/2μL, 10 μg/2μL) were applied to treat H. armigera, and the results showed that low concentrations of JHA had no significant effect on F1 generation survival of H. armigera, but the cumulative survival rate at 10 μg/2μL was 63% significantly lower than that of the control group. The total number of eggs laid female adult increased first, then decreased with JHA concentrations increasing. The results showed that in insect, the effects of JHA on different physiological processes had different threshold (Chen, Reference Chen2013). Alternatively, JHAMT protein expression was significantly reduced by RNAi in Drosophila melanogaster, but there was no significant effect on its development (Niwa et al., Reference Niwa, Niimi, Honda, Yoshiyama, Itoyama, Kataoka and Shinoda2008). This may be related to individual differences, and different thresholds in different individuals (Tibbetts et al., Reference Tibbetts, Izzo and Tinghitella2011). In addition, A. gossypii belongs to r-strategy insect which are achieved by a distinctive life-history strategy consisting of rapid development, early reproduction and a short life cycle when the aphids were treated with dsJHAMT. The rescue experiment showed that juvenile hormone analogues (methoprene) did rescue the rapid growth of A. gossypii caused by silencing AgJHAMT, and its whole life cycle was almost coinciding with that of the sprayed H2O control group after methoprene rescue. Similar findings have been reported in other studies. For example, in L. decemlineata, feeding on dsJHAMT1 and dsJHAMT2 caused 2nd instar and 4th instar mortality, and the JH analogue pyriproxyfen rescued the negative performance (Fu et al., Reference Fu, Li, Zhou, Meng, Lu, Guo and Li2016). Knockdown of TcMT3 in T. castaneum larvae resulted in precocious larval-pupal metamorphosis, which was rescued by methoprene (Minakuchi et al., Reference Minakuchi, Namiki, Yoshiyama and Shinoda2008). These results indicated that AgJHAMT silencing promoted the development of cotton aphids, and illuminated the function of AgJHAMT in the developmental period of cotton aphids.
To sum up, we obtained the differentially expressed gene AgJHAMT, which was enriched in the hormone synthesis pathway related to the growth and development of cotton aphids by transcriptome analysis. These results of the AgJHAMT expression pattern, AgJHAMT silencing and JHA rescuing experiments elucidated that the AgJHAMT gene played an important role in the developmental period of nymphs. These results not only implied that low expression of AgJHAMT would accelerate development of cotton aphids to a certain extent, but also provided insights into the molecular mechanism of the TG cotton delayed the development of A. gossypii.
Supplementary material
Transcriptome data validation (Figure. S1); Cumulative mortality and cumulative reproduction of A. gossypii after spraying dsJHAMT. (Figure. S2). Date control statistics of transcriptome of A. gossypii (Table S1); Primer sequences (Table S2). The supplementary material for this article can be found at https://doi.org/10.1017/S000748532400049X.
Acknowledgements
This work was supported by the Natural Science Foundation of Xinjiang Uygur Autonomous Region in China (2022D01D07), the National Natural Science Foundation of China-Xinjiang Joint Fund, Training Program of Local Excellent Youth Scholars (U1603331) and Graduate Student Research and Innovation Projects in Xinjiang Autonomous Region (XJ2023G031).
Authors’ contributions
Lianjun Zhang: Conceptualisation, Data curation, Methodology, Formal analysis, Writing - original draft, Writing - review & editing, Funding acquisition. Yuan Li: Methodology, Validation. Xinhui Xu: Formal analysis, Validation. Mengmeng Feng: Methodology. Rukiya Turak: Methodology. Xiaoning Liu: Conceptualisation, Project administration, Resources, Validation, Supervision, Writing - review & editing, Funding acquisition. Hongsheng Pan: Writing - review & editing. All authors read and approved the manuscript.
Competing interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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